DNA Barcoding in the Genome Era

Ya-ping ZhangKunming Institute of Zoology

Chinese Academy of Sciences

Yunnan University

The Second International Barcoding of Life Conference(16-21 September 2007, Taipei,

Taiwan)

Sinocyclocheilus grahami

Challenge: loss of biodiversity and expert

Known biodiversity 1.7million named species of plants and animals

Estimated biodiversity 10 million species

How many named species can be identified by experts now and future?

How many experts will survive in future?

2002 First proposal of “DNA Taxonomy” by Tautz et al2003 First proposal of a DNA barcoding system based on Mitochondrial Cytochrome C Oxidase Subunit 1 (COI) sequence diversity by Hebert et alMar, 2003 A meeting titled “Taxonomy and DNA” in Cold Spring Harbor, USA

Jul, 2003 Accession to the website “http:www.barcodeoflife.org/”Sep, 2003 A meeting titled “Taxonomy, DNA and the Barcode of Life” in Cold Spring Harbor, USA

DNA Barcoding

May, 2004 Establishment of Consortium for the Barcode of Life, CBOL

Feb, 2005 The First International Barcode Conference in London

Total Barcode Records

Kingdoms of Life Barcoded

Total Barcode Records 303,359http://www.boldsystems.org/views/login.php

Formally Described Species With Barcodes 31,763

Barcoding in China

NSFC ?

MOST?National Basic Research Program of China (973 Program)

My laboratory

animal DNA bank

Diagnosis of described species and Discovery of new species

Plant Barcoding(nuclear ITS, Plastid trnH-psbA intergenic sp

acer, rbcL)(flowering plants) (land plants)

Fungi and Algae Barcoding(mitochondrial COI)

(Penicillium: 0.06% intraspecific and 5.6% interspecific divergence)

(Alaria: 1% vs. 2.2-4.7%) (Red Macroalgae: 0.5% vs. 4.5-13.6%)

Fungi Barcoding

Algae Barcoding

Vertebrates Barcoding

Invertebrates Barcoding

(mitochondrial COI)

(amphibians: 7-14% intraspecific)

(birds: 0.43% intraspecific and 7.93% interspecific)

(moths: 0.25% vs. 6.5%)

(fish: 0.39% vs. 9.93%)

(ants: 8.51% interspecific)

(spiders: 1.4% vs. 16.4%)

(mayflies: 1% vs. 18%)

(primates: 0.3% vs. 5.88%)

(Lepidoptera: 0.17-0.46% vs. 4.41-6.02%)

Animal Barcoding

……

……

Delimiting Cryptic Species

Can we build a bridge between BOL and TOL in genome era?

Goal?

Technology?

Cost? 10 million species

Hajibabaei et al. 2007

Vences et al. 2005

Phylogenetic signals

DNA Barcoding for Australia’s fish species: Phylogenetic signals for four major clusters

DNA Barcoding for Big-headed flies: Phylogenetic signals for four main species groups ……

Should CBOL recommend additional mitochondrial DNA fragments?

Nuclear Pseudogenes

Phylogenetic signal

Root vs tip?

Can we harvest without too much additional investment?

An assessment of candidate mt barcoding genes by phylogenetic analysis of complete mitochondrial genome sequences

In Genome Era: Taking advantage of the completely sequenced genomes

Ursidae (bear) family as a test case

The bear family Ursidae includes eight species and has been suggested to be classified into three genera: Ailuropoda (giant panda), Tremarctos (spectacled bear), and Ursus (brown, polar, sloth, sun, and Asiatic and American black bears)

The family Ursidae represents a typical example of rapid evolutionary radiation and recent speciation events, dating back to mid-Miocene about 20 million years ago.

Analyzing the complete mt genome sequences from all eight closely related bear species not only provides a robust Ursidae phylogenetic framework but evaluates the evolutionary rates and performance of individual mtDNA genes

Analyses give the rough appearance of relatively superior performance of CO1, ND5, ND4, CYTB, 16SrRNA, and ND2 genes

Recommendation?

Introgression Nuclear PseudogenesIdentifying Chinese Leporids (Lagomorphs, Leporidae, Lepus)Chinese leporids underwent an early and recent radiation. Members of the Chinese leporids are characterized by similar morphological characters that contribute to the problematic taxonomy of the groupChinese leporids are generally classified into nine species based on morphometric features: L. timidus (moutain hare) L. sinensi (Chinese hare) L. Mandschuricus (Manchurian hare) L. melainus (Manchurian black hare) L. capensis (Cape hare) L. oiostolus (Woolly hare) L. yarkandensis (Yarkand hare) L. hainanus (Hainan hare) L. comus (Yunnan hare)

0.02

L. oiostolus

L. comus

L. hainanus

L.timidus DAXINGANLING

84

100

97

L.Capensis SHANXIL.Capensis SHANXI

L.Capensis QINGHAIL.Capensis QINGHAIL.sinensis GANSU

L.sinensis GANSUL.sinensis GANSUL.Capensis GANSU

L.Capensis SICHUANL.sinensis GANSU

L.sinensis GANSUL.timidus XINJIANG

L.timidus XINJIANGL.timidus XINJIANG

L.mandschuricus HEILONGJIANGL.melainus JILINL.mandschuricus JILIN

L.timidus HAERBINL.mandschuricus JILIN

7594

L.Yarkandensis XINJIANGL.Yarkandensis XINJIANGL.Yarkandensis XINJIANG

L.Yarkandensis XINJIANGL.Yarkandensis XINJIANGL.Yarkandensis XINJIANG

L.Yarkandensis XINJIANGL.Yarkandensis XINJIANGL.Yarkandensis XINJIANGL.Yarkandensis XINJIANG

L.Yarkandensis XINJIANGL.Yarkandensis XINJIANG

89

76

L.timidus JILINL.timidus HAERBINL. Mandschuricus DALIAN

100

L.sinensis GUIYANGL.Capensis SHANDONGL.sinensis FUJIANL.sinensis HUNANL.Capensis SHANDONGL.Sinensis GUIYANGL.Capensis SHANDONGL.Capensis SHANDONGL.Capensis SHANDONGL.Capensis SHANDONG

L. hainanus HAINANDAOL.. europaeus L. hainanus HAINANDAOL. hainanus HAINANDAOL. hainanus HAINANDAOL. hainanus HAINANDAOL. hainanus HAINANDAOL. hainanus HAINANDAOL. hainanus HAINANDAO

100

75

87

100

L. oiostolus SICHUANL. oiostolus XIZANGL. oiostolus SICHUANL. comus YUNNANL. comus YUNNAN

L. comus YUNNANL. comus YUNNANL. comus YUNNAN

L. comus YUNNAN

L. comus YUNNAN

O. cuniculus O. collaris

60

9999

86

L. yarkandensis

L. sinensis and L. capensis mixed

L. sinensis and L. capensis mixed

L. timidus, L. mandschuricus, and L.melainus mixed

L. timidus, L. mandschuricus, and L.melainus mixed

COI (648bp; unpublished data)

DNA barcords reveal

(1) coamplification of NUMTS in six individuals of Chinese leporids

(3) non-monophyletic species in phylogenetic tree: L.sinensis and L.capensis

(2) Intraspecific divergence within L. capensis and L. sinensis were 4% and 4.5%, which were much larger than intraspecific divergence.

Mt introgression

The real challenge of identification lies with closely allied species, species pairs with very recent origins, and hybridsUsing some level of mtDNA divergence as a yardstick for species boundaries is difficult for situations: (1) in between species and subspecies, species and populations, (2) overlap of intraspecific and interspecific divergence values

0.1

mulatta 5

mulatta 6

cyclopis

mulatta 1

mulatta 3

mulatta 4

mulatta 2

1.0

1.0

1.0 1.00.93

0.95

Papio

sylvanus

arctoides 6

arctoides 3

arctoides 1,2,5

arctoides 4

fascicularis 4

fascicularis 1,2

fascicularis 3

leonina 4

silenus 2

0.66 1.0

0.960.99

1.01.0

1.0

1.0

0.9

assamensis 5

assamensis 9

assamensis 11

assamensis 7

assamensis 3

assamensis 2

thibetana 1

thibetana 2,3,4

thibetana 5

assamensis 4

assamensis 8,10

assamensis 6

assamensis 1

assamensis 12

1.0

1.0

1.0

0.89

1.0

1.0

1.0

1.0

1.0

1.0

1.0

0.99

leonina 3

leonina 2

leonina 1

silenus 1

1.0 1.0

1.0

0.81

0.55

Papio

97

sylvanus

leonina 4

leonina 2

leonina 1

leonina 3

silenus 1

silenus 2

96

100

100

100

96

76

98

53

100

mulatta 6

mulatta 5 100

98

cyclopis

mulatta 3

mulatta 1 100

mulatta 4

mulatta 252

52

arctoides 1,2,5

arctoides 6

arctoides 3

100

100fascicularis 4

fascicularis 1,2

fascicularis 3100

58

arctoides 4

100

76

assamensis 3

assamensis 4

assamensis 6

assamensis 1

assamensis 12

assamensis 2thibetana 1

thibetana 5

thibetana 2,3,4

56

assamensis 8,1081

56100

82

62

69

assamensis 5

assamensis 9

assamensis 11

assamensis 7

10071

100

Sylvanus group

fasc

icul

aris

gro

upsi

nic

a g

roup

sile

nu

s

grou

p

BI tree MP tree

Species classification should be changed?

DNA divergence is not sufficient as a sole criterion for delineating species

DNA sequence information with traditional means, e.g. morphological and ecological data

“type sequences” (i.e. a reference framework for testing species status) for barcode?

Morphologically well-defined

Geographically well-known

What kind of samples should be included for investigating species limits?

Avoiding overestimate or underestimate the DNA divergence threshold values to identify candidate speciesPhylogeny or sequence difference?

Species: sister species pairs?

Subspecies?

Populations?

Individuals?

Recommendation for sample selection?

Do we need new sequencing technology for barcoding?

What kind?

Biodiversity assessment with high throughput technology?

Acknowledgement

Dr. Li Yu

Yu-wei Li

Jiang Liu

Dr. Qing-Qing Li

Dr. Li-ping He

Thank you for your attention!